The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β

Abstract

Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.

Figure 1

Effects of collagen gel and TGF-β on actin cytoskeleton in MSCs. MSCs were grown on collagen-coated culture dishes (stiff substrates) or collagen gel for 1 day in the absence or presence of TGF-β. The cells were stained for F-actin by using phalloidin (A-D) or stained for SM α-actin (E-H). Scale bar=50 μm.

Figure 2

Effects of collagen gel and TGF-β on gene expression and MSC differentiation. (A) MSCs were cultured on collagen-coated stiff substrates or collagen gel for 2 days in the absence or presence of TGF-β. Cells were lysed for qPCR analysis. * indicates significant difference (P<0.05). # indicates significant difference from the gene expression in MSCs on stiff substrate without TGF-β (P<0.05). § indicates significant difference from the gene expression in MSCs on stiff substrate with TGF-β (P<0.05). (B-E) Differentiation of MSCs into SMC lineage. MSCs were cultured on collagen-coated stiff substrates or collagen gel for 2 weeks in the absence or presence of TGF-β. Cells were double-stained for SM α-actin (red) and calponin-1 (green). (F-I) Under the same culture condition as in B-E, GAGs were stained by Alcian Blue. Scale Bars are 100 μm.

Figure 3

Effect of collagen gel on MSC differentiation into adipogenic lineage. (A) MSCs were cultured on collagen-coated stiff substrates or collagen gel for 2 days in the absence or presence of TGF-β. Cells were lysed for qPCR analysis as described in Figure 2A. (B-E) Adipogenic differentiation of MSCs. MSCs were cultured on collagen-coated stiff substrates or collagen gel for 2 weeks in the absence or presence of adipogenic medium. Lipids were stained by Oil Red. Scale Bar=50 μm.

Figure 4

Modulation of MSC proliferation and differentiation by matrix stiffness. (A) Stiffness of PA gels using 6% acrylamide and different bis-acrylamide concentrations. (B) MSCs were grown on PA gel for 1 day. Proliferation of MSCs on PA gels was quantified by BrdU incorporation. The number of cells in S phase for each sample was normalized to that on 15 kPa substrate to show fold-changes. * indicates significant difference (P<0.05). (C-E) Phalloidin staining of F-actin after 1-day culture. (F) MSCs were grown on collagen-coated stiff substrates or PA gels for 2 days in the absence or presence of TGF-β, and lysed for qPCR analysis. *, # and § indicate significant differences as in Figure 2A.

Figure 5

Effects of Rho activation on the gene expression in MSCs on stiff and soft substrates. (A) MSCs were cultured for 1 day and lysed for Rho activity assay followed by immunoblotting analysis. (B) MSCs with the expression of GFP or GFP-RhoA(v14) were cultured for 2 days and lysed for qPCR analysis. * indicates significant difference (P<0.05). # indicates significant difference from GFP-MSCs on stiff substrates (P<0.05). § indicates significant difference from GFP-MSCs on soft substrates (P<0.05).

Figure 6

Effect of Rho activation on actin cytoskeleton and the expression and assembly of α-actin. MSCs with the expression of GFP or GFP-RhoA(v14) were cultured on collagen-coated stiff substrates or PA gels for 1 day. (A-D) MSCs were stained for actin filaments by using phalloidin. (E-H) MSCs were stained for SM α-actin. Scale bar=50 μm.

Figure 7

Cell adhesion strength mediated the effects of matrix stiffness. (A-B) MSCs were cultured collagen-coated stiff substrates or PA gels for 1 day and stained for vinculin. Scale bar=50 μm. (C) MSCs were subjected to shear stress and the percentage of cells remaining on the substrates was quantified. * indicates significant difference from all other samples. (D-F) MSCs were treated with a blocking antibody against integrin β1 (anti-β1) or a control IgG, seeded on collagen-coated culture dishes, and either stained for actin filaments (green) and vinculin (red) (D-E) or lysed for qPCR analysis (F). Scale bar=50 μm. * indicates significant difference (P<0.05).